3-Phosphoinositide-dependent protein kinase-1 activates the peroxisome proliferator-activated receptor-γ and promotes adipocyte differentiation

Adipocyte differentiation is regulated largely through the actions of the peroxisome proliferator-activated receptor ( PPAR)gamma nuclear receptor and the insulin signaling pathway. 3-Phosphoinositidedependent protein kinase-1 (PDK1) serves as a critical regulatory point in insulin signaling through its ability to phosphorylate the activation loop of several protein kinase families. The present study was undertaken to determine the interrelationships between the PDK1 and PPAR gamma signaling pathways, and their association with adipocyte differentiation. Coexpression of PDK1 and PPAR gamma 1 in 293T cells stimulated PPAR gamma response element-dependent reporter gene activity in either the presence or absence of ligand. PDK1-mediated stimulation of PPAR gamma 1 activity was comparable in magnitude to the coactivator activated in breast cancer-1, and was blocked by either the corepressor silencing mediator of retinoid and thyroid hormone receptor or dominant-negative PAX8-PPAR gamma 1 . Heterologous Gal4-PPAR gamma 1 assays indicated that PDK1 interacted with the ligand binding domain, and physically associated with PPAR gamma 1; however, PDK1-mediated stimulation was not dependent on phosphorylation of PPAR gamma 1 by PDK1. PDK1 stimulatory activity was eliminated by mutation of the gamma-helical hydrophobic motifs in PDK1, L268XII, and V313XXLL, and expression of the gamma-helical region encompassing these motifs stimulated PPAR gamma response element-dependent transcription. PDK1-PPAR gamma interaction was confirmed by chromatin immunoprecipitation analysis of the lipoprotein lipase and adipocyte fatty acid-binding protein promoters. In cells expressing PDK1 and PPAR gamma, binding to PPAR gamma response elements occurred, which was enhanced by treatment with a PPAR gamma agonist. Expression of PDK1 in 3T3-L1 or COMMA-1D mammary epithelial cells promoted adipocyte differentiation in the presence of a PPAR gamma agonist that was comparable to the response of PPAR gamma 1-transfected cells in the presence of agonist; expression of PDK1 and PPAR gamma resulted in a synergistic effect. Adipocyte differentiation in the presence of a PPAR gamma agonist was markedly attenuated in PDK1 null cells. These results suggest that PDK1 can function as a PPAR gamma 1 coactivator independently of its catalytic activity and establishes an important mechanistic link between adipocyte differentiation and the insulin signaling pathway.